N. Matsumori, M. Murata et al.
tion (not corrected for dilution with Triton X-100 solution) to that before
the addition of Triton X-100 (%OD) was then calculated.
level. The force-field parameters for the Chol portion were taken from
reference [36]. For all simulations and energy minimizations GROMACS
(Ver. 3.3.1) modeling software was used. All lipids and Chol bond lengths
were constrained with the LINCS algorithm,[38] whereas the SETTLE al-
gorithm[39] was used for water. The simulations were performed in the
constant NPT (the number of molecules, the pressure, and the tempera-
ture) ensemble with semi-isotropic pressure coupling. The time step was
set to 2.0 fs. Each simulation covered a time scale of 10.0 ns, which in-
cluded an equilibration period of 8.0 ns prior to the analysis step. The
treatment of long-range interactions were handled by using the particle-
Fluorescence quenching experiments: By the procedure described above,
the following multilamellar vesicles (50 nmol total lipids) were prepared
in PBS buffer (pH 7, 1 mL): SM/Chol/12SLPC (1:1:1) containing 1%
DPH (F sample); SM/Chol/12SLPC (1:1:1) (Fb sample); DOPC/Chol/
12SLPC (1:1:1) containing 1% DPH (Fc sample); DOPC/Chol/12SLPC
(1:1:1) (Fbc sample); SM/Chol/DOPC (1:1:1) containing 1% DPH (F0
sample); SM/Chol/DOPC (1:1:1) (F0b sample); DOPC/Chol (2:1) con-
taining 1% DPH (F0c sample); DOPC/Chol (2:1) (F0bc sample). To evalu-
ate domain formation of the conjugates in this assay, 1:1 SM-Chol mix-
ture was replaced by each conjugate. Fluorescence was measured on a
JASCO FP-6600 spectrofluorometer at RT with a 1 cm excitation, 4 mm
emission path length quartz cuvette. The excitation/emission wavelength
settings were 359 and 427 nm, respectively. Excitation and emission slits
with a band pass of 1 and 6 nm, respectively, were used for all measure-
ments. DF/Fo values were calculated by inputting the fluorescence intensi-
mesh Ewald technique.[40]
A 1.0 nm cutoff in the direct space and
0.15 nm Fourier spacing were used. For the van der Waals interactions, a
twin range cut-off (1.0/1.6 nm) was applied. The neighbor-pair list was
updated every five steps. Temperature boundary conditions were set by
using the Nose–Hoover thermostat[41] with a time constant (t)=0.1 ps.
Pressure-boundary conditions were set with the Parrinello–Rahman baro-
stat,[42] t=1.0 ps. The reference temperature was T=323 K, which is
above the main phase-transition temperature of 18:0 SM.
ties
(FcÀFbc)/
into
ACHTUNGTRENNUNG
the
following
equation:
DF/Fo =(FÀFb)/(F0ÀF0b)À
G
Fluorescence anisotropy measurements: Multilamellar vesicles of lipids
containing DPH (2 mol%) were prepared by drying a solution of SM
(160 nmol), 1:1 SM-Chol (total 160 nmol), conjugate 1or 2 (80 nmol) and
DPH (3.2 nmol) in 1:1 MeOH/CHCl3 under a stream of argon, redissolv-
ing the residue in CHCl3, and drying under a stream of argon. After the
lipids were further dried under high vacuum for at least 12 h, they were
hydrated (swelled) by addition of PBS buffer (pH 7.0, 3 mL). To uniform-
ly disperse the lipids and form homogeneous multilamellar lipid vesicles,
each sample was vigorously vortexed at 658C, well above the phase-tran-
sition temperature of SM, and then cooled to RT. The samples were sub-
jected to three freeze–thaw cycles in liquid nitrogen and a water bath
maintained at 658C. Fluorescence polarization measurements were per-
formed with a JASCO FP-6600 spectrofluorometer equipped with a
JASCO ADP-303 polarization accessory. Quartz cuvettes with a path
length of 1 cm were used. The excitation wavelength was set at 358 nm
and emission was monitored at 430 nm. Excitation and emission slits with
a band pass of 1 and 10 nm, respectively, were used for all measurements.
The excitation slit used was the smallest possible to minimize any photo-
Acknowledgements
We are grateful to Dr. Jun Shimokawa and Prof. Tohru Fukuyama (The
University of Tokyo) for helpful discussion on the nosyl chemistry. This
work was supported by Grant-In-Aids for Scientific Research (B) (No.
20310132) and (S) (No. 18101010) from MEXT, Japan.
[11] For a recent review, see P. S. Niemelꢂ, M. T. Hyvçnen, I. Vattulai-
[12] a) N. Matsumori, N. Eiraku, S. Matsuoka, T. Oishi, M. Murata, T.
mori, Y. Umegawa, S. Matsuoka, H. Ueno, H. Ikeuchi, T. Oishi, M.
ACHTUNGTRENNUNGisomerization of DPH during irradiation. The measurement temperature
was raised from 30 to 708C with a 28CminÀ1 gradient, and fluorescence
was measured with a 2 s response. Polarization values were calculated
with Spectra Manager software attached to the spectrofluorometer.
Atomic-scale simulation: We studied three bilayers composed of:
1) 64 SM and 64 Chol units, 2) 64 molecules of 1, and 3) 64 molecules of
2. The initial structure of the membrane comprised of 64 SM and 64 Chol
units was built by modification of the membrane composed of 128 SM
and 3655 water molecules, reported by Niemelꢂ et al.,[35] 64 SM molecules
were chosen from 128 SM molecules of the membrane and replaced by
64 Chol molecules. The same number of SM molecules were replaced in
each leaflet and the structure of the bilayer was energy minimized.
Force-field parameters for the SM and Chol molecules were taken from
references [35,36], respectively. The initial structure of Chol was also
taken from reference [36]. The initial structures of the membranes com-
posed from conjugates 1 and 2 were built as follows: the molecular struc-
tures of the conjugates were generated by Dundee PRODRG Server,[37]
then the Chol parts were replaced by the reported structure.[36] A leaflet
was formed by distributing 32 molecules on the x,y plane to avoid van
der Waals contacts. The second layer was obtained from the first by 1808
rotation. The obtained bilayer was hydrated with 3655 simple point
charge water molecules and energy minimization was performed to give
the initial membrane structure. Force-field parameters for conjugate mol-
ecules were constructed in three parts for SM, the linker, and Chol. For
the SM unit, the bonded and nonbonded parameters were taken from
reference [35], expect that the parameters of ammonium were adopted
from GROMOS force field (ff43Ga1.itp). For the linker, the bonded pa-
rameters were taken from GROMOS force field (ff43Ga1.itp) but the
[13] J. Aittoniemi, P. S. Niemelꢂ, M. T. Hyvçnen, M. Karttunen, I. Vattu-
[17] M. P. Veiga, J. L. R. Arrondo, F. M. Goni, A. Alonso, D. Marsh, Bio-
[18] T. Yamamoto, H. Hasegawa, T. Hakogi, S. Katsumura, Org. Lett.
[19] P. Shenbagamurthi, B. Kundu, J. M. Becker, P. Naider, Int. J. Peptide
Protein Res. 1985, 25, 187–196.
[22] C. E. Dreef, C. J. J. Elie, P. Hoogerhout, G. A. van der Marel, J. H.
À
À
À
bonded parameters for the O NH structure were adopted from the
parameters generated by using the Dundee PRODRG Server. The par-
[24] C. J. J. Elie, C. E. Dreef, R. Verduyn, G. A. Van Der Marel, J. H. van
À
À
À
tial charge for the O NH structure was calculated at the HF/6–31G
8574
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 8568 – 8575